Therapeutic antibodies have become an important therapeutic modality in drug development. New technologies in antibody engineering enable manifold possibilities in antibody design like alterations in valency, specificity and affinity resulting in a great spectrum of different antibodies. In order to identify and select antibodies, a large number of potential therapeutics has to be evaluated to identify promising candidates during early drug development. For this reason, there is a great interest in fast and efficient production of those recombinant proteins for research and early drug development.
Antibodies are complex biomolecules, requiring posttranslational modifications like glycosylation and correct folding to gain full bioactivity, and therefore, mammalian cells are the major employed cells for recombinant production. There are two methods to produce antibodies in these cells: stable and transient gene expression. Stable gene expression is a very effective way to produce recombinant antibodies because the encoding nucleic acids integrate into the genome of the host cell, what enables their continuous expression. However, the generation of a cell line stably expressing the antibody is very labor- and time-consuming. Hence, transient gene expression is the method of choice in early drug development, as it offers the solution for the need of rapidly producing the material necessary. Albeit gene expression is not continuous with this method, it is possible to produce the recombinant antibody within a few days.
Valproic Acid (VPA) is a short chain fatty acid used in the clinic as an anticonvulsive agent and mood-stabilizing drug (Blaheta et al.; 2005). It is mostly applied to treat epilepsy, bipolar disorders or migraine headache. Since VPA inhibits proliferation, induces apoptosis and induces differentiation of a variety of tumor cells, it is often implicated in the treatment of cancer. Recent studies identified several additional cellular pathways affected by VPA, like PKC inhibition, Wnt-signaling activation and ERK activation (Fujiki et al.; 2013). Another interesting feature of VPA is its ability to inhibit histone deacetylases (HDACs) (Gottlicher et al.; 2001).
Valproic acid (VPA) is commonly used for transient gene expression (TGE) enhancement. As histone inhibitory activity was discovered for VPA it was proposed that its effect on TGE is mediated by inhibiting the deacetylation of histone tails. This hypothesis arose because it is assumed that transiently transfected plasmids integrate into the chromatin structure and therefore underlie epigenetic regulation of gene expression by histone modification.
However, VPA treatment also decreases viable cell density (VCD) and cell viability of the cells. Therefore, VPA was shown to induce not only cell cycle arrest but subsequently also the extrinsic as well as the intrinsic apoptotic pathway, respectively (Xu et al.; 2007). Thus, combined treatment of VPA with caspase inhibitors significantly prevented apoptotic cell death induced by VPA (Han et al.; 2013).
Backliwal, G., et al. (Biotechnol. Bioeng. (2008)) reported that valproic acid is a viable alternative to sodium butyrate for enhancing protein expression in mammalian cell culture. It is suggested herein that the effect of valproic acid is based on a histone deacetylase inhibition (HDACi).
It was shown in various cell lines including human embryonic kidney (HEK) 293 cells that VPA directly inhibits histone deacetylases (HDACs) (Gottlicher, Minucci et al. 2001). Confirming this observation, hyperacetylation of Histone H4 was observed after VPA treatment (Phiel, Zhang et al. 2001).
In US 2009/023186 the use of valproic acid for enhancing production of recombinant proteins in mammalian cells is reported.
In WO 2015/000624 a method for producing antibodies using ovine b-cells and uses thereof is reported.
In WO 2010/050903 chimeric Flagellins for vaccines are reported.
In WO 2015/036583 the combination of epigenetic factors and bispecific compounds targeting CD33 and CD3 in the treatment of myeloid leukemia is reported.
In WO 2014/022758 a single agent anti-PD-L1 and PD-L2 dual binding antibodies and methods of use is reported.
R. Furumai reported FK228 (Depsipeptide) as a natural prodrug that inhibits class I histone deacetylases (Cancer Res. 62 (2002) 4916).